טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentIbrahim Marjieh
SubjectBacterial Capture by Oligo-Acyl-Lysines
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Mor Amram
Full Thesis textFull thesis text - English Version


Abstract

Towards the development of improved tools for rapid, sensitive and reliable bacterial detection, some new approaches have used peptides or proteins to target bacterial surface components.  For instance, antimicrobial peptides (AMP) are being developed for various applications including microbial detection offering broad spectrum of bacterial recognition and simple structure. 

Here we propose the use of oligomers of acylated lysines, synthetic mimics of AMPs, composed of tandem repeats of amide linked fatty acids and lysines, offering improved stability and simpler synthesis compared to natural AMP. Thus, the resin-linked oligomers of acylated lysine (ROAK) concept is shown to achieve efficient, broad-spectrum and rapid bacterial capture. We describe the capture of three medically relevant bacterial species (E. coli, K. pneumoniae and P. aeruginosa) on the K-7α12 ROAK (K, lysyl; α12, tandem repeat of lauryl-lysyl), under both incubation and continuous flow conditions. Such capture was sustained for different ROAK derivatives generated during a structure-activity relationship study. This led to an improved ROAK, as shorter derivatives (i.e. K-3α12, composed of 7 residues instead of 15 for the initial ROAK) were shown to have similar capture attributes yet better bacterial recovery yield towards further exploitation.

Another aspect of this study, involves an attempt to improve bacterial detection sensitivity. We show conditions under which significant proportions of the captured bacteria can be eluted - live or dead - for bacterial enrichment purposes prior to identification or quantification of bacterial load in samples by qPCR. Namely, K-3α12 based columns were shown to achieve a significant enrichment of bacteria in accordance with initial sample volume, allowing a 20 fold increase in bacterial counts compared to initial counts for samples of 100 mL.

Collectively, the data provide strong support for a robust and potentially useful application of the ROAK approach in bacterial capture/analysis from tap water and possibly from more complex media.